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Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

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PARP1 protein controls Cajal body integrity.The following antibodies were used for immunostainings: Guinea Pig anti-Coilin (green); rabbit anti-Fibrillarin (red); and the DNA binding dye Draq5 (blue). N – nucleolus. The position of CB is indicated with arrow. Wild-type (A) and PARP1 mutant (B, C, D) salivary glands were used for immunostaining. E. Immunoprecipitation assays using mouse anti-Coilin antibody. The total protein extracts from wild-type (WT) and PARP1 mutant (PARP−) flies were used. To detect protein on Western blots, rabbit anti-Coilin, rabbit anti-Fibrillarin, and mouse anti-Actin antibodies were used.
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pgen-1000387-g002: PARP1 protein controls Cajal body integrity.The following antibodies were used for immunostainings: Guinea Pig anti-Coilin (green); rabbit anti-Fibrillarin (red); and the DNA binding dye Draq5 (blue). N – nucleolus. The position of CB is indicated with arrow. Wild-type (A) and PARP1 mutant (B, C, D) salivary glands were used for immunostaining. E. Immunoprecipitation assays using mouse anti-Coilin antibody. The total protein extracts from wild-type (WT) and PARP1 mutant (PARP−) flies were used. To detect protein on Western blots, rabbit anti-Coilin, rabbit anti-Fibrillarin, and mouse anti-Actin antibodies were used.

Mentions: Previously, we demonstrated that PARP1 protein is involved in chromatin loosening and transcriptional activation as well as the establishment and maintenance of nucleolus [6]. Nucleolar and CB proteins, such as Fibrillarin, relocated from nuclei to cytoplasm in PARP1 mutant cells [6]. In order to test whether PARP1 protein is required for CB function, we compared localization of typical CB resident proteins, Fibrillarin and Coilin, in wild-type flies and PARP1 mutants. In wild-type animals, Coilin protein localizes exclusively in CB (Figure 2A). Fibrillarin protein occupies CB as well as nucleoli (Figure 2A). Fibrillarin and Coilin completely co-localized in the CB (Figure 2A) and demonstrated physical interaction in co-immunoprecipitation experiments (Figure 2E). Drosophila PARP1 mutants survive until maternally-contributed Parp1 mRNA and PARP1 protein degrade [6]. The earliest phenotype which we detected in those mutants is the relocation of Fibrillarin from CBs (Figure 2B). At the same time, Fibrillarin still occupies the nucleolus, and CB is detected as a single intact organelle (Figure 2B). At the later stages, however, we detected the breakdown of single CB into disintegrating Coilin-positive particles (Figure 2C). Finally, even at the most severe PARP1 mutant stage, when Fibrillarin relocalized from nucleolus to cytoplasm, we were still able to identify a few small, separated Coilin-containing bodies in nuclei (Figure 2D). Co-immunoprecipitation experiments using antibodies against Coilin confirmed the absence of interaction between Coilin and Fibrillarin in the PARP1 mutant animals (Figure 2E). We conclude from these data that PARP1 protein is necessary for interaction of key protein components of CB and may, therefore, also be essential for CB.


Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

PARP1 protein controls Cajal body integrity.The following antibodies were used for immunostainings: Guinea Pig anti-Coilin (green); rabbit anti-Fibrillarin (red); and the DNA binding dye Draq5 (blue). N – nucleolus. The position of CB is indicated with arrow. Wild-type (A) and PARP1 mutant (B, C, D) salivary glands were used for immunostaining. E. Immunoprecipitation assays using mouse anti-Coilin antibody. The total protein extracts from wild-type (WT) and PARP1 mutant (PARP−) flies were used. To detect protein on Western blots, rabbit anti-Coilin, rabbit anti-Fibrillarin, and mouse anti-Actin antibodies were used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637609&req=5

pgen-1000387-g002: PARP1 protein controls Cajal body integrity.The following antibodies were used for immunostainings: Guinea Pig anti-Coilin (green); rabbit anti-Fibrillarin (red); and the DNA binding dye Draq5 (blue). N – nucleolus. The position of CB is indicated with arrow. Wild-type (A) and PARP1 mutant (B, C, D) salivary glands were used for immunostaining. E. Immunoprecipitation assays using mouse anti-Coilin antibody. The total protein extracts from wild-type (WT) and PARP1 mutant (PARP−) flies were used. To detect protein on Western blots, rabbit anti-Coilin, rabbit anti-Fibrillarin, and mouse anti-Actin antibodies were used.
Mentions: Previously, we demonstrated that PARP1 protein is involved in chromatin loosening and transcriptional activation as well as the establishment and maintenance of nucleolus [6]. Nucleolar and CB proteins, such as Fibrillarin, relocated from nuclei to cytoplasm in PARP1 mutant cells [6]. In order to test whether PARP1 protein is required for CB function, we compared localization of typical CB resident proteins, Fibrillarin and Coilin, in wild-type flies and PARP1 mutants. In wild-type animals, Coilin protein localizes exclusively in CB (Figure 2A). Fibrillarin protein occupies CB as well as nucleoli (Figure 2A). Fibrillarin and Coilin completely co-localized in the CB (Figure 2A) and demonstrated physical interaction in co-immunoprecipitation experiments (Figure 2E). Drosophila PARP1 mutants survive until maternally-contributed Parp1 mRNA and PARP1 protein degrade [6]. The earliest phenotype which we detected in those mutants is the relocation of Fibrillarin from CBs (Figure 2B). At the same time, Fibrillarin still occupies the nucleolus, and CB is detected as a single intact organelle (Figure 2B). At the later stages, however, we detected the breakdown of single CB into disintegrating Coilin-positive particles (Figure 2C). Finally, even at the most severe PARP1 mutant stage, when Fibrillarin relocalized from nucleolus to cytoplasm, we were still able to identify a few small, separated Coilin-containing bodies in nuclei (Figure 2D). Co-immunoprecipitation experiments using antibodies against Coilin confirmed the absence of interaction between Coilin and Fibrillarin in the PARP1 mutant animals (Figure 2E). We conclude from these data that PARP1 protein is necessary for interaction of key protein components of CB and may, therefore, also be essential for CB.

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

Show MeSH
Related in: MedlinePlus